bioRxiv preprint doi: https://doi.org/10.1101/2019.12.18.881649; this version posted December 19, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

1 Integrase-RNA interactions underscore the critical role of integrase in HIV-1 virion

2 morphogenesis

3

4 Short title: Key role for HIV-1 integrase-RNA interactions in virion maturation

5

6 Jennifer Elliott1, Jenna E. Eschbach1, Pratibha C. Koneru2, Wen Li3,4, Maritza Puray

7 Chavez1, Dana Townsend1, Dana Lawson1, Alan N. Engelman3,4, Mamuka Kvaratskhelia2,

8 Sebla B. Kutluay1

9

10 1 Department of Molecular Microbiology, Washington University School of Medicine, Saint Louis,

11 MO 63110, USA

12 2 Division of Infectious Diseases, University of Colorado School of Medicine, Aurora, CO 80045

13 3 Department of Cancer Immunology and Virology, Dana-Farber Cancer Institute, Boston, MA

14 02215

15 4 Department of Medicine, Harvard Medical School, Boston, MA 02115

16

17

18 Correspondence: [email protected]

19

20

21

22

23

24

25

26 bioRxiv preprint doi: https://doi.org/10.1101/2019.12.18.881649; this version posted December 19, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

27 ABSTRACT

28 A large number of HIV-1 integrase (IN) alterations, referred to as class II substitutions, exhibit

29 pleotropic effects during virus replication. However, the underlying mechanism for the class II

30 phenotype is not known. Here we demonstrate that all tested class II IN substitutions

31 compromised IN-RNA binding in virions by one of three distinct mechanisms: i) markedly

32 reducing IN levels thus precluding formation of IN complexes with viral RNA; ii) adversely

33 affecting functional IN multimerization and consequently impairing IN binding to viral RNA; iii)

34 directly compromising IN-RNA interactions without substantially affecting IN levels or functional

35 IN multimerization. Inhibition of IN-RNA interactions resulted in mislocalization of the viral

36 ribonucleoprotein complexes outside the capsid lattice, which led to premature degradation of

37 the viral genome and IN in target cells. Collectively, our studies uncover causal mechanisms for

38 the class II phenotype and highlight an essential role of IN-RNA interactions for accurate virion

39 maturation.

40

41 INTRODUCTION

42 Infectious HIV-1 virions are formed in a multistep process coordinated by interactions

43 between the HIV-1 Gag and Gag-Pol polyproteins, and the viral RNA (vRNA) genome. At the

44 plasma membrane of an infected cell, Gag and Gag-Pol molecules assemble around a vRNA

45 dimer and bud from the cell as a spherical immature virion, in which the Gag proteins are

46 radially arranged [1-3]. As the immature virion buds, the viral protease enzyme is activated and

47 cleaves Gag and Gag-Pol into their constituent domains triggering virion maturation [1, 2].

48 During maturation the cleaved nucleocapsid (NC) domain of Gag condenses with the RNA

49 genome and pol-encoded viral enzymes [reverse transcriptase (RT) and integrase (IN)] inside

50 the conical capsid lattice, composed of the cleaved capsid (CA) protein, which together form the

51 core [1-3].

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52 After infection of a target cell, RT in the confines of the reverse transcription

53 complex (RTC) synthesizes linear double stranded DNA from vRNA [4]. The vDNA is

54 subsequently imported into the nucleus, where the IN enzyme catalyzes its insertion into the

55 host cell chromosome [5, 6]. Integration is mediated by the intasome nucleoprotein complex that

56 consists of a multimer of IN engaging both ends of linear vDNA [7]. While the number of IN

57 protomers required for intasome function varies across Retroviridae, single particle cryogenic

58 electron microscopy (cryo-EM) structures of HIV-1 and Maedi-visna virus indicate that lentivirus

59 integration proceeds via respective higher-order dodecamer and hexadecamer IN arrangements

60 [8, 9], though a lower-order intasome comprised of an HIV-1 IN tetramer was also resolvable by

61 cryo-EM [9].

62 A number of IN substitutions which specifically arrest HIV-1 replication at the integration

63 step have been described [10]. These substitutions are grouped into class I to delineate them

64 from a variety of other IN substitutions, which exhibit pleiotropic effects and are collectively

65 referred to as class II substitutions [10-12]. Class II IN substitutions or deletion of entire IN

66 impair proper particle assembly [11, 13-25], morphogenesis [11, 15, 21-23, 26-28] and reverse

67 transcription in target cells [10, 11, 17, 19-21, 23, 25-44], in some cases without impacting IN

68 catalytic function [15, 16, 19, 20, 30, 31, 34, 36, 45-47]. A hallmark morphological defect of

69 these viruses is the formation of aberrant viral particles with viral ribonucleoprotein (vRNP)

70 complexes mislocalized outside of the conical CA lattice [11, 15, 21-23, 26-28]. Strikingly similar

71 morphological defects are observed in virions produced from cells treated with allosteric

72 integrase inhibitors (ALLINIs, also known as LEDGINs, NCINIs, INLAIs or MINIs) [26, 27, 48-

73 55]. ALLINIs induce aberrant IN multimerization in virions by engaging the V-shaped pocket at

74 the IN dimer interface, which also provides a principal binding site for the host integration

75 targeting cofactor lens epithelium-derived growth factor (LEDGF)/p75 [50, 54, 56-60]. The

76 recent discovery that HIV-1 IN binds to the vRNA genome in virions and that inhibiting IN-RNA

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77 interactions leads to the formation of eccentric particles provided initial clues about the role of IN

78 during virion morphogenesis [28].

79 HIV-1 IN consists of three independently folded protein domains: the N-terminal domain

80 (NTD), catalytic core domain (CCD), and C-terminal domain (CTD) [7, 61], and vRNA binding is

81 mediated by a constellation of basic residues within the CTD [28]. However, class II IN

82 substitutions are located throughout the entire length of the IN protein [10, 12], which raises the

83 question as to how these substitutions impair virus maturation. The structural basis for IN

84 binding to RNA is not yet known; however, in vitro evidence indicates that IN binds RNA as

85 lower-order multimers, and conversely RNA binding may prevent the formation of higher order

86 IN multimers [28]. Notably, aberrant IN multimerization underlies the inhibition of IN-RNA

87 interactions by ALLINIs [28] and subsequent defects in virion maturation [26-28, 48, 49, 51-55].

88 Therefore, it seems plausible that class II IN substitutions may exert their effect on virus

89 replication by adversely affecting functional IN multimerization. However, a systematical

90 evaluation of the effects of IN substitutions on IN multimerization, IN-RNA binding, and virion

91 morphology is lacking. As such, it remains an open question how functional IN multimerization

92 and/or IN-RNA interactions influence correct virion morphogenesis.

93 Eccentric virions generated via class II IN substitutions or ALLINI treatment are defective

94 for reverse transcription in target cells [10, 11, 17, 19-21, 23, 25-44, 48, 49, 51, 54, 58, 62]

95 despite containing equivalent levels of RT and vRNA genome as wild type (WT) particles [26,

96 63]. In addition, neither the condensation of the viral genome by NC [26, 63] nor its priming [63]

97 appear to be affected. We and others have recently shown that premature loss of the viral

98 genome and IN, as well as spatial separation of RT from vRNPs, may underlie the reverse

99 transcription defect observed in eccentric viruses generated in the presence of ALLINIs or the

100 class II IN R269A/K273A substitutions [59, 64]. These findings support a model in which the

101 capsid lattice or IN binding to vRNA itself is necessary to protect viral components from the host

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102 environment upon entering a target cell. Whether the premature loss of the viral genome and IN

103 is a universal outcome of other class II IN substitutions is unknown.

104 In this work, we aimed to determine the molecular basis of how class II IN substitutions

105 exert their effects on HIV-1 replication. In particular, by detailed characterization of how class II

106 substitutions impact IN multimerization, IN-RNA interactions and virion morphology, we aimed to

107 dissect whether loss of IN binding to vRNA or aberrant IN multimerization underlies the

108 pleiotropic defects observed in viruses bearing class II IN mutations. Remarkably, we found that

109 class II substitutions either prevented IN binding to the vRNA genome or precluded the

110 formation of IN-vRNA complexes through reducing or eliminating IN from virions. We show that

111 IN tetramers have a strikingly higher affinity towards vRNA than IN monomers or dimers, and a

112 large number of class II IN substitutions inhibited IN binding to RNA indirectly through

113 modulating functional IN tetramerization. In contrast, R262A/R263A and R269A/K273A

114 substitutions within the CTD and the K34A change within the NTD did not perturb IN tetramer

115 formation, and thus likely directly interfered with IN binding to RNA. Irrespective of how IN-RNA

116 binding was inhibited, all class II IN mutant viruses formed eccentric particles with vRNPs

117 mislocalized outside of the CA lattice. Subsequently, this led to premature loss of the vRNA

118 genome as well as IN, and spatial separation of RT and CA from the vRNPs in target cells.

119 Taken together, our findings uncover causal mechanisms for the class II phenotype and

120 highlight the essential role of IN-RNA interactions for the formation of correctly matured virions

121 and vRNP stability in HIV-1-infected cells.

122

123 MATERIALS AND METHODS 124 125 Plasmids

126 The pNLGP plasmid consisting of the HIV-1NL4-3 -derived Gag-Pol sequence inserted into the

127 pCR/V1 plasmid backbone [65] and the CCGW vector genome plasmid carrying a GFP reporter

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128 under the control of the CMV promoter [66, 67] were previously described. The pLR2P-vprIN

129 plasmid expressing a Vpr-IN fusion protein has also been previously described [68]. Mutations

130 in the IN coding sequence were introduced into both the pNLGP plasmid and the HIV-1NL4-3 full-

131 length proviral plasmid (pNL4-3) by overlap extension PCR. Briefly, forward and reverse primers

132 containing IN mutations in the pol reading frame were used in PCR reactions with antisense and

133 sense outer primers containing unique restriction endonuclease sites (AgeI-sense, NotI-

134 antisense for NLGP and AgeI-sense, EcoRI-antisense for pNL4-3), respectively. The resulting

135 fragments containing the desired mutations were mixed at 1:1 ratio and overlapped

136 subsequently using the sense and antisense primer pairs. The resulting fragments were

137 digested with the corresponding restriction endonucleases and cloned into pNLGP and pNL4-3

138 plasmids. IN mutations were introduced into the pLR2P-vprIN plasmid using the QuickChange

139 Site-Directed Mutagenesis kit (Agilent Technologies). Presence of the desired mutations and

140 absence of unwanted secondary changes were verified by Sanger sequencing.

141 Cells and viruses

142 HEK293T cells (ATCC CRL-11268) and HeLa-derived TZM-bl cells (NIH AIDS Reagent

143 Program) were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal

144 bovine serum. MT-4 cells were maintained in RPMI 1640 medium supplemented with 10% fetal

145 bovine serum. CHO K1-derived pgsA-745 cells (CRL-2242, ATCC) that lack a functional

146 xylosyltransferase enzyme and as a result do not produce glycosaminoglycans were maintained

147 in Dulbecco’s modified Eagle’s / F12 (1:1) media supplemented with 10% fetal bovine serum

148 and 1 mM L-glutamine. Single-cycle GFP reporter viruses pseudotyped with vesicular stomatitis

149 virus G protein (VSV-G) were produced by transfection of HEK293T cells with pNLGP-derived

150 plasmids, the CCGW vector genome carrying GFP, and VSV-G expression plasmid at a ratio of

151 5:5:1, respectively, using polyethyleneimine (PolySciences, Warrington, PA). Full-length viruses

152 pseudotyped with VSV-G were produced by transfecting HEK293T cells with the pNL4-3-

153 derived plasmids and VSV-G plasmid at a ratio of 4:1 (pNL4-3:VSV-G).

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154 Immunoblotting

155 Viral and cell lysates were resuspended in sodium dodecyl sulfate (SDS) sample buffer and

156 separated by electrophoresis on Bolt 4-12% Bis-Tris Plus gels (Life Technologies), blotted onto

157 nitrocellulose membranes and probed overnight at 4°C with the following antibodies in Odyssey

158 Blocking Buffer (LI-COR): mouse monoclonal anti-HIV p24 antibody (183-H12-5C, NIH AIDS

159 reagents), mouse monoclonal anti-HIV integrase antibody [69], rabbit polyclonal anti-HIV

160 integrase antibody raised in-house against Q44-LKGEAMHGQVD-C56 peptide and hence

161 unlikely to be affected by the substitutions introduced into IN in this study, rabbit polyclonal anti-

162 HIV-1 reverse transcriptase antibody (6195, NIH AIDS reagents), rabbit polyclonal anti-Vpr

163 antibody (11836, NIH AIDS Reagents), rabbit polyclonal anti-MA antibody (4811, NIH AIDS

164 Reagents). Membranes were probed with fluorophore-conjugated secondary antibodies (LI-

165 COR) and scanned using a LI-COR Odyssey system. IN and CA levels in virions were

166 quantified using Image Studio software (LI-COR). For analysis of the fates of core components

167 in infected cells, antibody incubations were done using 5% non-fat dry milk. Membranes were

168 probed with HRP-conjugated secondary antibodies and developed using SuperSignalTM West

169 Femto reagent (Thermo-Fisher).

170 Analysis of reverse transcription products in infected cells

171 MT-4 cells were grown in 24-well plates and infected with VSV-G pseudotyped pNL4-3 viruses

172 (either WT or class II IN mutant) at a multiplicity of infection (MOI) of 2 in the presence of

173 polybrene. Six h post-infection cells were collected, pelleted by brief centrifugation, and

174 resuspended in PBS. DNA was extracted from cells using the DNeasy Blood and Tissue Kit

175 (Qiagen) as per kit protocol. Quantity of HIV-1 vDNA was measured by Q-PCR using primers

176 specific for early reverse-transcripts.

177 Vpr-IN transcomplementation experiments

178 A class I IN mutant virus (HIV-1NL4-3 IND116N) was trans-complemented with class II mutant IN

179 proteins as described previously [68]. Briefly, HEK293T cells grown in 24-well plates were co-

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180 transfected with a derivative of the full-length HIV-1NL4-3 proviral plasmid bearing a class I IN

181 substitution (pNL4-3D116N), VSV-G, and derivatives of the pLR2P-vprIN plasmid bearing class II

182 IN mutations at a ratio of 6:1:3. Two days post-transfection cell-free virions were collected from

183 cell culture supernatants. Integration capability of the trans-complemented class II IN mutants

184 was tested by infecting MT-4 cells and measuring the yield of progeny virions in cell culture

185 supernatants over a 6-day period as described previously [68]. In brief, MT-4 cells were

186 incubated with virus inoculum in 96 V-bottom well plates for 4 h at 37°C after which the virus

187 inoculum was washed away and replaced with fresh media. Immediately following removal of

188 the virus inoculum and during the six subsequent days the quantity of virions present in the

189 culture supernatant was quantified by measuring RT activity using a Q-PCR-based assay [70].

190 CLIP experiments

191 CLIP experiments were conducted as previously described [28, 71, 72]. Cell-free HIV-1 virions

192 were isolated from transfected HEK293T cells. Briefly, cells in 15-cm cell culture plates were

193 transfected with 30 µg full-length proviral plasmid (pNL4-3) DNA containing the WT sequence or

194 indicated pol mutations within the IN coding sequence. Cells were grown in the presence of 4-

195 thiouridine for 16 h prior to virus harvest. Two days post transfection cell culture supernatants

196 were collected and filtered through 0.22 µm filters and pelleted by ultracentrifugation through a

197 20% sucrose cushion using a Beckman SW32-Ti rotor at 28,000 rpm for 1.5 h at 4°C. Virus

198 pellets were resuspended in phosphate-buffered saline (PBS) and UV-crosslinked. Following

199 lysis in RIPA buffer, IN-RNA complexes were immunoprecipitated using a mouse monoclonal

200 anti-IN antibody [69]. Bound RNA was end-labeled with γ-32P-ATP and T4 polynucleotide

201 kinase. The isolated protein-RNA complexes were separated by SDS-PAGE, transferred to

202 nitrocellulose membranes and exposed to autoradiography films to visualize RNA. Lysates and

203 immunoprecipitates were also analyzed by immunoblotting using antibodies against IN.

204 IN multimerization in virions

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205 HEK293T cells grown on 10-cm dishes were transfected with 10 µg pNL4-3 plasmid DNA

206 containing the WT sequence or indicated pol mutations within IN coding sequence. Two days

207 post-transfection cell-free virions collected from cell culture supernatants were pelleted by

208 ultracentrifugation through a 20% sucrose cushion using a Beckman SW41-Ti rotor at 28,000

209 rpm for 1.5 h at 4°C. Pelleted virions were resuspended in 1X PBS and treated with ethylene

210 glycol bis(succinimidyl succinate) (EGS) (ThermoFisher Scientific), a membrane permeable

211 crosslinker, at a concentration of 1 mM for 30 min at room temperature. Crosslinking was

212 stopped by addition of SDS sample buffer. Samples were subsequently separated on 3-8% Tris-

213 acetate gels and analyzed by immunoblotting using a mouse monoclonal anti-IN antibody [69].

214 Size exclusion chromatography (SEC)

215 All of the mutations were introduced into a plasmid backbone expressing His6 tagged pNL4-3-

216 derived IN by QuikChange site directed mutagenesis kit (Agilent) [60]. His6 tagged recombinant

217 pNL4-3 WT and mutant INs were expressed in BL21 (DE3) E. coli cells followed by nickel and

218 heparin column purification as described previously [60, 73]. Recombinant WT and mutant INs

219 were analyzed on Superdex 200 10/300 GL column (GE Healthcare) with running buffer

220 containing 20 mM HEPES (pH 7.5), 1 M NaCl, 10% glycerol and 5 mM BME at 0.3 mL/min flow

221 rate. The proteins were diluted to 10 µM with the running buffer and incubated for 1 h at 4°C

222 followed by centrifugation at 10,000g for 10 min. Multimeric form determination was based on

223 the standards including bovine thyroglobulin (670,000 Da), bovine gamma-globulin (158,000

224 Da), chicken ovalbumin (44,000 Da), horse myoglobin (17,000 Da) and vitamin B12 (1,350 Da).

225 Analysis of IN-RNA binding in vitro

226 Following SEC of IN as above, individual fractions of tetramer, dimer and monomer forms were

227 collected and their binding to TAR RNA was analyzed by an Alpha screen assay as described

228 previously [28]. Briefly, 100 nM His6 tagged IN fractions (tetramer, dimer and monomer) were

229 incubated with nickel acceptor beads while increasing concentrations of biotinylated-TAR RNA

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230 was incubated with streptavidin donor beads in buffer containing 100 mM NaCl, 1 mM MgCl2, 1

231 mM DTT, 1 mg/mL BSA, 25 mM Tris (pH 7.4). Followed by 2-h incubation at 4°C, they were

232 mixed and the reading was taken after 1 h incubation at 4°C by PerkinElmer Life Sciences

233 Enspire multimode plate reader. The Kd values were calculated using OriginLab software.

234 Virus production and transmission electron microscopy

235 Cell-free HIV-1 virions were isolated from transfected HEK293T cells. Briefly, cells grown in two

236 15-cm cell culture plates (107 cells per dish) were transfected with 30 µg full-length proviral

237 plasmid (pNL4-3) DNA containing the WT sequence or indicated pol mutations within IN coding

238 sequence using PolyJet DNA transfection reagent as recommended by the manufacturer

239 (SignaGen Laboratories). Two days after transfection, cell culture supernatants were filtered

240 through 0.22 µm filters and pelleted by ultracentrifugation using a Beckman SW32-Ti rotor at

241 26,000 rpm for 2 h at 4oC. Fixative (2.5% glutaraldehyde, 1.25% paraformaldehyde, 0.03%

242 picric acid, 0.1 M sodium cacodylate, pH 7.4) was gently added to resulting pellets, and samples

243 were incubated overnight at 4oC. The following steps were conducted at the Harvard Medical

244 School Electron Microscopy core facility. Samples were washed with 0.1 M sodium cacodylate,

245 pH 7.4 and postfixed with 1% osmium tetroxide /1.5% potassium ferrocyanide for 1 h, washed

246 twice with water, once with maleate buffer (MB), and incubated in 1% uranyl acetate in MB for 1

247 h. Samples washed twice with water were dehydrated in ethanol by subsequent 10 minute

248 incubations with 50%, 70%, 90%, and then twice with 100%. The samples were then placed in

249 propyleneoxide for 1 h and infiltrated overnight in a 1:1 mixture of propyleneoxide and TAAB

250 Epon (Marivac Canada Inc.). The following day the samples were embedded in TAAB Epon and

251 polymerized at 60 °C for 48 h. Ultrathin sections (about 60 nm) were cut on a Reichert Ultracut-

252 S microtome, transferred to copper grids stained with lead citrate, and examined in a JEOL

253 1200EX transmission electron microscope with images recorded on an AMT 2k CCD camera.

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254 Images were captured at 30,000x magnification, and over 100 viral particles per sample were

255 counted by visual inspection.

256 Equilibrium density sedimentation of virion core components in vitro

257 Equilibrium density sedimentation of virion core components was performed as previously

258 described [64]. Briefly, HEK293T cells grown in 10-cm cell culture plates were transfected with

259 10 µg pNLGP plasmid DNA containing the WT sequence or indicated pol mutations within IN

260 coding sequence. Two days post-transfection cell-free virions collected from cell culture

261 supernatants were pelleted by ultracentrifugation through a 20% sucrose cushion using a

262 Beckman SW41-Ti rotor at 28,000 rpm for 1.5 hr at 4°C. Pelleted viral-like particles were

263 resuspended in PBS and treated with 0.5% Triton X-100 for 2 min at room temperature.

264 Immediately after, samples were layered on top of 30-70% linear sucrose gradients prepared in

265 1X STE buffer (100 mM NaCl, 10 mM Tris-Cl (pH 8.0), 1 mM EDTA) and ultracentrifuged using

266 a Beckman SW55-Ti rotor at 28,500 rpm for 16 h at 4°C. Fractions (500 µL) collected from the

267 top of the gradients were analyzed for IN, CA, and MA by immunoblotting as detailed above.

268 Biochemical analysis of virion core components in infected cells

269 Biochemical analysis of retroviral cores in infected cells was performed as described previously

270 [74]. Briefly, pgsA-745 cells were infected with VSV-G pseudotyped single cycle GFP-reporter

271 viruses or its derivatives synchronously at 4°C. Following the removal of virus inoculum and

272 extensive washes with PBS, cells were incubated at 37°C for 2 h. To prevent loss of vRNA due

273 to reverse-transcription, cells were infected in the presence of 25 µM nevirapine. Post-nuclear

274 supernatants were separated by ultracentrifugation on 10-50% linear sucrose gradients using a

275 Beckman SW55-Ti rotor at 30,000 rpm for 1 h at 4°C. Ten 500 µl fractions from the top of the

276 gradient were collected, and CA, IN, and vRNA in each fraction were analyzed by either

277 immunoblotting or Q-PCR [74]. A SYBR-Green-based Q-PCR assay [70] was used to determine

278 RT activity in the collected sucrose fractions.

279 Visualization of vRNA in infected cells

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280 Viral RNA was visualized in infected cells according to the published multiplex

281 immunofluorescent cell-based detection of DNA, RNA and Protein (MICDDRP) protocol [75].

282 VSV-G pseudotyped HIV-1NL4-3 virus stocks were prepared as described above and

283 concentrated 40X using a lentivirus precipitation solution (ALSTEM). PgsA-745 cells were

284 plated on 1.5 mm collagen-treated coverslips (GG-12-1.5-Collagen, Neuvitro) placed in 24-well

285 plates one day prior to infection. Synchronized infections were performed by incubating pre-

286 chilled virus inoculum on the cells for 30 min at 4°C. Cells were infected with WT virus at a MOI

287 of 0.5, or with an equivalent number (normalized by RNA copy number) of IN mutant viral

288 particles. After removal of the virus inoculum cells were washed with PBS and either

289 immediately fixed with 4% paraformaldehyde, or incubated at 37°C for 2 h before fixing. To

290 prevent loss of vRNA due to reverse-transcription, cells were infected and incubated in the

291 presence of 25 µM nevirapine. Following fixation, cells were dehydrated with ethanol and stored

292 at -20°C. Prior to probing for vRNA, cells were rehydrated, incubated in 0.1% Tween in PBS for

293 10 min, and mounted on slides. Probing was performed using RNAScope probes and reagents

294 (Advanced Cell Diagnostics). Briefly, coverslips were treated with protease solution for 15 min in

295 a humidified HybEZ oven (Advanced Cell Diagnostics) at 40 °C. The coverslips were then

296 washed with PBS and pre-designed anti-sense probes [75] specific for HIV-1 vRNA were

297 applied and allowed to hybridize with the samples in a humidified HybEZ oven at 40 °C for 2 h.

298 The probes were visualized by hybridizing with preamplifiers, amplifiers, and finally, a

299 fluorescent label. First, pre-amplifier 1 (Amp 1-FL) was hybridized to its cognate probe for 30

300 min in a humidified HybEZ oven at 40 °C. Samples were then subsequently incubated with Amp

301 2-FL, Amp 3-FL, and Amp 4A-FL for 15 min, 30 min, and 15 min respectively. Between adding

302 amplifiers, the coverslips were washed with a proprietary wash buffer. Nuclei were stained with

303 DAPI diluted in PBS at room temperature for 5 min. Finally, coverslips were washed in PBST

304 followed by PBS and then mounted on slides using Prolong Gold Antifade.

305 Microscopy and image quantification

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306 Images were taken using a Zeiss LSM 880 Airyscan confocal microscope equipped with a

307 ×63/1.4 oil-immersion objective using the Airyscan super-resolution mode. 10 images were

308 taken for each sample using the ×63 objective. Numbers of nuclei and vRNA punctae in images

309 were counted using Volocity software (Quorum Technologies). The number of vRNA punctae

310 per 100 nuclei were recorded at 0 h post-infection (hpi) and 2 hpi for each virus, and the number

311 at 2 hpi compared to the number at 0 hpi.

312 Analysis of the fate of vRNA genome in MT4 cells

313 MT-4 cells were infected with VSV-G pseudotyped HIV-1 NL4-3 WT or equivalent number of

314 mutant viruses (normalized by RT activity) synchronously at 4°C. After removal of virus

315 inoculum and extensive washes with PBS, cells were incubated at 37°C for 6 h in the presence

316 of 25 µM nevirapine. Immediately after synchronization (0 h) and at 2 and 6 h post-infection

317 samples were taken from the infected cultures and RNA was isolated using TRIzol Reagent.

318 The amount of viral genomic RNA was measured by Q-RT-PCR.

319

320 RESULTS

321 Characterization of the replication defects of class II IN mutant viruses

322 Substitutions in IN that exhibited a class II phenotype (i.e. assembly, maturation or

323 reverse transcription defects [10-44, 76, 77] or affected IN multimerization [46, 78-81] were

324 selected from past literature. The location of these substitutions depicted on the cryo-EM

325 structure of the tetrameric HIV-1 intasome complex [9] indicate that many are positioned at or

326 near monomer-monomer or dimer-dimer interfaces (Fig. 1A-B). While not apparent in the

327 tetrameric intasome complex, the CTD mediates IN tetramer-tetramer interactions in the higher-

328 order dodecamer IN structure [9] and has also been shown to mediate IN multimerization in vitro

329 [15].

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330 IN mutations were introduced into the replication competent pNL4-3 molecular clone and

331 HEK293T cells were transfected with the resulting plasmids. Cell lysates and cell-free virions

332 were subsequently analyzed for Gag/Gag-Pol expression, processing, particle release and

333 infectivity. While substitutions in IN had no measurable effect on Gag (Pr55) expression, modest

334 effects on Gag processing in cells was visible for several missense mutant viruses including

335 H12N, N18I, K34A, Y99A, K103E, W108R, F185K, Q214L/Q216L, L242A, V260E, as well as

336 the ΔIN mutant (Fig. 2A). Nevertheless, particle release was largely similar between WT and IN

337 mutant viruses, as evident by the similar levels of CA protein present in cell culture supernatants

338 (Fig. 2A, lower panels).

339 Three distinct phenotypes became apparent by assessing the amount of virion-

340 associated IN and RT enzymes (Fig. 2A and Fig. S1A). First, virion-associated IN was at least

341 5-fold less than WT with several mutants, including H12N, N18I, K103E, W108R, F185K,

342 L242A, and V260E (Fig. 2A and Table 1). Notably, these substitutions also reduced levels of

343 Gag-Pol processing intermediates in producer cells (Fig. 2A) and RT in virions (Fig. 2A and Fig.

344 S1A), suggesting that they likely destabilized the Gag-Pol precursor. Near complete lack of

345 processing intermediates with the K14A and N18I substitutions, despite the presence of fully

346 processed RT and IN in virions (detected using a separate polyclonal antibody), is likely due to

347 inaccessibility of epitopes recognized by the monoclonal anti-IN antibody in the processing

348 intermediates. Second, the R228A substitution abolished full-length IN in virions without

349 impacting cell- or virion-associated Gag-Pol levels or processing intermediates; however, a

350 faster migrating species due to altered charge in IN or that may represent product of aberrant IN

351 processing and/or IN degradation was visible. A similar but more modest defect was observed

352 for the K34A mutant, which was incorporated into virions at a modestly reduced level alongside

353 a smaller protein species. Third, the remainder of the IN substitutions did not appear to affect IN

354 or Gag-Pol levels in cells or virions.

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355 With the exception of E96A, nearly all of the IN substitutions reduced virus titers at least

356 100-fold compared to the WT (Fig. 2B), which corresponded with reduced levels of reverse-

357 transcription in infected cells (Fig. 2C). In line with previous reports [19, 20, 34], class II mutant

358 IN molecules had variable levels of catalytic activity as assessed by the ability of Vpr-IN proteins

359 to transcomplement a catalytically inactive IN (D116N, [11, 45]) in infected cells [68, 82]. All

360 Vpr-IN fusion proteins, except for the H12N mutant which likely decreased the stability of the

361 Vpr-IN fusion protein, were expressed at similar levels in cells (Fig. S1B). We found that K14A,

362 E96A, Y99A, K103A, V165A, R187A, K188E R199A, K236E, and R269A/R273A IN mutants

363 trans-complemented a catalytically inactive IN at levels similar to the WT, whereas W108R,

364 R228A, and V260E mutants were unable to do so (Fig. 2D-E). The inability of W108R, R228A,

365 and V260E mutants to transcomplement implies that they are impaired for integration, a result in

366 line with previous observations [46, 81]. The remainder of the IN mutants restored integration,

367 albeit at significantly lower than WT levels (Fig. 2D-E). These results suggest that the majority of

368 the class II mutant INs retain structural integrity and at least partial catalytic activity in the

369 presence of a complementing IN protein. Cumulatively, these data show that some class II

370 substitutions in IN can affect the stability and/or processing of virion associated proteins, but

371 they all universally lead to the formation of non-infectious virions that are blocked at reverse

372 transcription in target cells, a hallmark of class II IN substitutions [10, 12].

373 Class II IN mutants abolish IN binding to RNA

374 Using complementary in vitro and CLIP-based approaches, we have previously shown

375 that IN interacts with the viral genome through multiple basic residues (i.e. K264, K266, R269,

376 K273) in its CTD [28]. In addition, IN-RNA interactions could also depend on proper IN

377 multimerization, as ALLINI-induced aberrant IN multimerization potently inhibited the ability of IN

378 to bind RNA [28]. Based on this, in the next set of experiments, we aimed to determine whether

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379 class II IN mutants bind vRNA, and if not, whether improper IN multimerization may underlie this

380 defect.

381 IN-vRNA complexes were immunoprecipitated from UV-crosslinked virions and the

382 levels of coimmunoprecipitating vRNA was assessed. Note that substitutions that significantly

383 reduced the amount of IN in virions (Fig. 2A, Table 1) were excluded from these experiments.

384 All class II IN mutant viruses contained similar levels of vRNA, ruling out any inadvertent effects

385 of the alterations on RNA packaging (Fig. 3A). While the catalytically inactive IN D116N bound

386 vRNA at a level that was comparable to the WT, nearly all of the class II IN mutant proteins

387 failed to bind vRNA (Fig. 3B). The E96A substitution, which had a fairly modest effect on virus

388 titers as compared to other IN mutants (Fig. 2B), decreased but did not abolish the ability of IN

389 to bind RNA (Fig. 3B). Thus, lack of RNA binding ability is a surprisingly common property of a

390 disperse set of class II IN mutants, despite the fact that many of the altered amino acid residues

391 are distally located from the CTD.

392 IN multimerization plays a key role in RNA binding

393 As it seemed unlikely that all of the class II IN substitutions directly inhibited IN binding to

394 RNA, we reasoned that they might indirectly abolish binding by perturbing proper IN

395 multimerization. To test whether class II IN substitutions altered IN multimerization in a relevant

396 setting, purified HIV-1NL4-3 virions were treated with ethylene glycol bis (succinimidyl succinate)

397 (EGS) to covalently crosslink IN in situ and virus lysates were analyzed by immunoblotting. IN

398 species that migrated at molecular weights consistent with those of monomers, dimers, trimers

399 and tetramers were readily distinguished in WT virions (Fig. 4A). In the majority of the class II

400 mutant particles, IN appeared to be predominantly monomeric, with little dimers and no readily

401 detectable tetramers (Fig. 4A). In contrast to this general pattern, K34A, E96A, R262A/R263A

402 and R269A/K273A IN mutants formed dimers and tetramers at similar levels to the WT (Fig.

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403 4A). An undefined smear was present at higher molecular weights for all virions, possibly as a

404 result of the formation of large IN aggregates upon cross-linking (Fig. 4A).

405 To corroborate these findings, we analyzed the multimerization properties of

406 recombinant WT, K34A, E96A, K188E, K236A and R262A/R263A IN proteins by SEC (Fig. 4B).

407 In line with the crosslinking studies in virions, WT, K34A and R262A/R263A IN molecules all

408 formed tetramers, while the levels of dimers varied between the mutants. For example, while IN

409 R262A/R263A presented similar levels of tetramers and dimers, IN K34A was primarily

410 tetrameric with a minor dimeric species, as evident by the broad right shoulder of the tetrameric

411 SEC peak (Fig. 4B). In contrast, E96A and K188E IN molecules almost exclusively formed

412 dimers and monomers with little evidence for tetramer formation (Fig. 4B). While K236E IN was

413 predominantly dimeric, the broad base of its chromatogram revealed some evidence for

414 tetramers and monomers as well (Fig. 4B).

415 We next determined the ability of IN monomers, dimers and tetramers to bind RNA to

416 test whether there is a causal link between the multimerization defects of class II IN

417 substitutions and RNA binding. Following size-exclusion chromatography (SEC)-based

418 separation of monomeric, dimeric and tetrameric IN, their affinity for TAR RNA, which

419 constitutes a high affinity binding site for IN [28], was assessed by an Alpha-screen assay.

420 Remarkably, while WT IN tetramers bound to TAR RNA at high affinity (2.68 ± 0.16 nM), neither

421 IN dimers nor monomers showed evidence of binding (Fig. 4C). Although IN K34A and IN

422 R262A/R263A could both form tetramers, IN K34A showed a reduced affinity for RNA while IN

423 R262A/R263A could not bind RNA at all (Fig. 4D).

424 Collectively, these results pointed to a key role of IN tetramerization in RNA binding and

425 suggest that a defect in proper multimerization underlies the inability of the majority of class II IN

426 mutants to bind vRNA. As the K34A and R262A/R263A substitutions did not affect IN

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427 tetramerization, our findings suggest that these residues may be directly involved in IN binding

428 to RNA.

429 Class II IN substitutions generate virions with eccentric morphology

430 We next sought to determine how preclusion or inhibition of IN-vRNA interactions

431 correlated with particle morphology. Virion morphology of a subset of the IN mutants that

432 inhibited vRNA interactions by three different mechanisms; i.e. those that decreased IN levels in

433 virions (N18I and W108R), those that may have directly inhibited IN binding to RNA (K34A,

434 R262A/R263A), and those that primarily altered IN multimerization (E87A, E96A, F185K,

435 R187A, L241A, L242A), was assessed by transmission electron microscopy (TEM). As

436 expected, the majority of WT particles contained an electron dense condensate representing

437 vRNPs inside the CA lattice, whereas an ΔRT-IN deletion mutant virus produced similar levels

438 of immature particles and eccentric particles (Fig. 5A-B). Remarkably, irrespective of how IN-

439 RNA interactions were inhibited, 70-80% of nearly all class II IN mutant particles exhibited an

440 eccentric morphology (Fig. 5A-B). Of note, the E96A mutant tended to produce less eccentric

441 and more mature particles than the other IN mutants. Because IN E96A retained partial binding

442 to vRNA in virions (Fig. 3B) and partial infectivity (Fig. 2B), we conclude that this infection-

443 deferred mutant harbors a partial class II phenotype.

444 Next, we tested whether inhibition of IN-RNA interactions through class II substitutions

445 changes the localization of IN in virions. The premise for this is based on our previous finding

446 that disruption of IN binding to vRNA through the IN R269A/K273A substitution leads to

447 separation of a fraction of IN from dense vRNPs and CA containing complexes [64]. Thus, we

448 predicted that inhibition of IN-RNA interactions through the above class II substitutions could

449 lead to a similar outcome. To this end, WT or class II IN mutant virions stripped of the viral lipid

450 envelope by brief detergent treatment were separated on sucrose gradients, and resulting

451 fractions were analyzed for CA, IN, and matrix (MA) content by immunoblotting [64, 83]. As

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452 before [64], WT IN migrated primarily in dense fractions, whereas the R269A/K273A mutant

453 migrated bimodally (Fig. 6A, B). In contrast to our hypothesis, the majority of IN mutants

454 sedimented similarly to WT IN and settled in the denser gradient fractions (Fig. 6A, B).

455 Exceptions were the K34A and R262A/R263A IN mutants, a fraction of which migrated in

456 soluble fractions similar to the R269A/K273A mutant, suggesting their localization outside of the

457 capsid lattice. None of the IN substitutions affected the migration pattern of CA (Fig. 6C), which

458 distributed bimodally between the soluble and dense fractions, nor the distribution of MA (data

459 not shown), which was found in mainly the soluble fractions. These results suggested that, with

460 the exception of the K34A, R262A/R263A, and R269A/K273A, IN mutant proteins may remain

461 associated with the CA lattice despite inhibition of IN-vRNA interactions.

462 Premature loss of vRNA and IN from class II IN mutant viruses upon infection of target

463 cells

464 We have previously shown that vRNA and IN are prematurely lost from cells infected

465 with the R269A/K273A class II IN mutant [64]. Given that eccentric vRNP localization is a

466 common feature of class II IN mutant viruses (Fig. 5), we next asked whether loss of vRNA in

467 target cells is a common outcome for other class II IN mutant viruses. As the majority of mutant

468 IN molecules appeared to remain associated with higher-order CA in virions (Fig. 6), we also

469 wanted to test whether they would be protected from premature degradation in infected cells.

470 The fates of viral core components in target cells were tracked using a previously

471 described biochemical assay [74]. For these experiments we utilized pgsA-745 cells (pgsA),

472 which lack surface glycosaminoglycans, and likely as a result can be very efficiently infected by

473 VSV-G-pseudotyped viruses in a synchronized fashion. PgsA cells were infected with WT or IN

474 mutant viruses bearing substitutions that inhibited IN-vRNA interactions directly and led to

475 mislocalization of IN in virions (i.e. K34A, R262A/R263A, R269A/K273A) or indirectly through

476 aberrant IN multimerization and did not appear to grossly affect IN localization in virions (i.e.

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477 E87A, V165A) (Fig 7A). Following infection, post-nuclear lysates were separated on linear

478 sucrose gradients, and fractions collected from gradients were analyzed for viral proteins (CA,

479 IN, RT) and vRNA by immunoblotting and Q-PCR-based assays, respectively.

480 As previously reported [64, 74], in cells infected with WT viruses, IN, RT, vRNA and a

481 fraction of CA comigrated in sucrose fractions 6-8, representing active RTCs (Fig. 7B-E). Note

482 that a large fraction of CA migrated in the top two soluble sucrose fractions representing CA that

483 had dissociated from the core as a result of uncoating or CA that was packaged into virions but

484 not incorporated into the capsid lattice [84, 85]. Notably, in cells infected with class II IN mutant

485 viruses, equivalent levels of CA (Fig. 7B) and RT (Fig. 7D) remained in the denser fractions,

486 whereas IN (Fig. 7C) and vRNA (Fig. 7E) were substantially reduced. Loss of vRNA and IN from

487 dense fractions, without any corresponding increase in the top fractions containing soluble

488 proteins and RNA, suggest their premature degradation and/or mislocalization in infected cells.

489 We next employed a complementary microcopy-based assay [75] in the context of full-

490 length viruses to corroborate these findings. Advantages of this approach over biochemical

491 fractionation experiments include the ability to track HIV-1 vRNA at the single-cell level with a

492 high degree of specificity (Fig. S2A), determine its subcellular localization, and to side-step

493 possible post-processing artifacts associated with biochemical fractionation. Cells were

494 synchronously infected with VSV-G pseudotyped HIV-1NL4-3 in the presence of nevirapine to

495 prevent vRNA loss due to reverse transcription, and vRNA levels associated with cells

496 immediately following synchronization (0 h) and 2 h post-infection were evaluated [75]. In WT-

497 infected cells, vRNA was clearly visible immediately after infection (Fig. 7F). Two h post

498 infection, cell associated vRNA had fallen to 60-80% of starting levels (Fig. 7F, S2C), likely as

499 the result of some viruses failing to enter or perhaps being degraded after entry. However, a

500 significant proportion of vRNA was still readily detectable. In contrast, in cells infected with the

501 IN mutant viruses the reduction in vRNA was greater, and by 2 h post-infection only 30-40%

502 remained (Fig. 7F and Fig. S2B-C). These results support the conclusion from the biochemical

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503 fractionation experiments that vRNA is prematurely lost from cells infected with class II IN

504 mutant viruses.

505 Finally, we tested whether our findings held true in physiologically relevant human cells.

506 MT-4 T cells were synchronously infected with WT or class II IN mutant VSV-G pseudotyped

507 HIV-1NL4-3 in the presence of nevirapine. Cells were collected immediately after synchronization

508 (0 h), 2 and 6 h post-infection, and the quantity of vRNA measured by Q-PCR. In line with the

509 above findings, vRNA levels decreased at a faster rate with the class II IN mutants as compared

510 to WT viruses, with half as much cell-associated vRNA remaining at 2 and 6 h post-infection for

511 the class II IN mutants (Fig. 7G). Treating cells with ammonium chloride to prevent fusion of the

512 VSV-G pseudotyped viruses rescued vRNA loss, and vRNA from WT and mutant viruses were

513 retained at equal levels, indicating that the loss of vRNA is dependent on entry into the target

514 cell (Fig. S2D). These findings agree with the previous experiments and demonstrate that class

515 II IN substitutions lead to the premature loss of vRNA genome also in human T cells.

516 DISCUSSION

517 Our findings highlight the critical role of IN-vRNA interactions in virion morphogenesis

518 and provide the mechanistic basis for how diverse class II IN substitutions lead to similar

519 morphological and reverse transcription defects. We propose that class II IN substitutions lead

520 to the formation of eccentric particles through three distinct mechanisms (Fig. 8): (i) depletion of

521 IN from virions thus precluding the formation of IN-vRNA complexes; ii) impairment of functional

522 IN multimerization and as a result, indirect disruption of IN-vRNA binding; iii) direct disruption of

523 IN-vRNA binding without substantially affecting IN levels or its inherent multimerization

524 properties. Irrespective of how IN binding to vRNA is inhibited, all substitutions led to the

525 formation of eccentric viruses that were subsequently blocked at reverse transcription in target

526 cells. We provide evidence that premature degradation of the exposed vRNPs and separation of

527 RT from the vRNPs underlies the reverse transcription defect of class II IN mutants (Fig. 7).

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528 Taken together, our findings cement the view that IN binding to RNA accounts for the role of IN

529 in accurate particle maturation and provide the mechanistic basis of why these viruses are

530 blocked at reverse transcription in target cells.

531 In regard to the first case (i) above, it was previously shown that IN deletion leads to the

532 formation of eccentric particles [11, 27]. Thus, it is reasonable to assume that missense

533 mutations that decreased IN levels in virions phenocopy IN deletion viruses. While it is also

534 possible that these substitutions additionally affected IN binding to vRNA or multimerization, we

535 could not reliably address these possibilities due to the extremely low levels of these proteins in

536 virions.

537 Our results show the striking affinity of IN tetramers to bind RNA compared with IN

538 monomers and dimers (Fig. 4C). In support of tetramerization being a prerequisite for RNA-

539 binding, the inability of a number of class II IN mutant proteins to bind RNA was accompanied

540 by a clear multimerization defect both in virions (Fig. 4A) and in vitro (Fig. 4B). The structural

541 basis for IN binding to RNA is not yet known; however, these findings are in line with the

542 previous in vitro evidence that hinted a link between IN multimerization and RNA-binding. For

543 example, IN binds RNA as lower-order multimers, and conversely RNA binding may inhibit the

544 formation of higher order IN multimers in vitro [28]. Notably, formation of open IN polymers that

545 occlude the IN CTD from RNA binding may underlie the inhibition of IN-RNA interactions by

546 ALLINIs [28, 58].

547 Based on MS-based footprinting experiments in vitro, we previously found that positively

548 charged residues within the CTD of IN (i.e. K264, K266, K273) directly contact RNA, as was

549 also validated by CLIP experiments [28]. Our findings here suggest that IN-vRNA contacts may

550 extend to nearby basic residues within the CTD, such as R262 and R263, and perhaps more

551 surprisingly, K34 within the IN NTD, as alterations of these residues did not prevent IN

552 tetramerization (Fig. 4A-B) but completely abolished IN-vRNA binding in virions (Fig. 3B) and

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553 reduced RNA-binding in vitro (Fig 4D). This raises the possibility of a second RNA-binding site

554 in the IN NTD. Structural analysis of IN in complex with RNA will be essential to definitively

555 determine how IN binds RNA as well as the precise multimeric species required for binding.

556 The mechanism by which IN-vRNA interactions mediate the encapsidation of vRNPs

557 inside the CA lattice remains unknown. One possibility is that the temporal coordination of

558 proteolytic cleavage events during maturation is influenced by IN-vRNA interactions [86, 87]. In

559 this scenario, the assembly of the CA lattice may become out of sync with the compaction of

560 vRNA by NC. Another possibility is that IN-vRNA complexes nucleate the assembly of the CA

561 lattice, perhaps by directly binding to CA. Notably, the biochemical assays performed herein

562 show that class II IN substitutions do not appear to affect the assembly and stability of the CA

563 lattice in vitro and in target cells. Although this finding is in disagreement with the previously

564 observed morphological aberrations of the CA lattice present in eccentric particles [26], it is

565 possible that the biochemical experiments used herein lack the level of sensitivity required to

566 quantitatively assess these aberrations. Further studies deciphering the crosstalk between IN-

567 RNA interactions and CA assembly will be critical to our understanding of the role of IN in

568 accurate virion maturation.

569 While the mislocalization of the vRNA genome in eccentric particles can be accurately

570 assessed by TEM analysis, precisely where IN is located in eccentric particles remains an open

571 question. Earlier studies based on biochemical separation of core components from detergent-

572 treated IN R269A/K273A virions indicated that IN may also mislocalize outside the CA lattice

573 [64]. In this study, only two class II IN mutants (K34A and R262A/R263A) revealed this

574 phenotype (Fig. 6A, B). It is intriguing that the bimodal distribution of IN in this experimental

575 setting was only seen with IN mutants that directly inhibited IN binding to vRNA. A possible

576 explanation for these observations is that improperly multimerized IN is retained within the CA

577 lattice or in association with it. Despite this co-migration pattern with CA, we found that both

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578 E87A and V165A mutant INs were rapidly lost in infected cells, suggesting that they are not fully

579 protected by CA upon cellular entry (Fig. 7C).

580 Why is the unprotected vRNA and IN prematurely lost in target cells? It seems evident

581 that the protection afforded by the CA lattice matters the most for vRNP stability, though we

582 cannot rule out that IN binding to vRNA may in and of itself stabilize both the genome and IN.

583 Alternatively, the AU-rich nucleotide content of HIV-1 may destabilize its RNA [88-90], similar to

584 several cellular mRNAs that encode for cytokines and growth factors [91]. Finally, RNA nicking

585 and deadenylation in virions by virion associated enzymes [92-94] may predispose retroviral

586 genomes to degradation when they are prematurely exposed to the cytosolic milieu. While

587 cytosolic IN undergoes proteasomal degradation when expressed alone in cells [95-99], we

588 have found previously that proteasome inhibition does not rescue vRNA or IN in target cells

589 infected with a class II IN mutant [64]. Further studies are needed to determine whether a

590 specific cellular mechanism or an inherent instability of vRNPs is responsible from the loss of

591 vRNA in infected cells.

592 In conclusion, we have identified IN-vRNA binding as the underlying factor for the role of

593 IN in virion morphogenesis and show that virion morphogenesis is necessary to prevent the

594 premature loss of vRNA and IN early in the HIV-1 lifecycle. Despite relatively high barriers,

595 drugs that inhibit the catalytic activity of IN do select for resistance, and additional drug classes

596 that inhibit IN activity through novel mechanisms of action would be a valuable addition to

597 currently available treatments. The finding that IN-vRNA interaction can be inhibited in multiple

598 ways- by directly altering residues in the IN CTD or by altering IN multimerization in virions- can

599 help guide the design of future anti-retroviral compounds.

600

601 FIGURE LEGENDS

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602 Figure 1. Class II IN substitutions locate throughout IN and cluster at interfaces that

603 mediate IN multimerization.

604 (A) Location of class II IN substitutions used in this study displayed in red on a single IN

605 monomer within the context of the HIV-1 IN tetramer intasome structure consisting of a dimer of

606 dimers (PDB 5U1C). The two dimers are displayed in either gray or green, with individual

607 monomers within each displayed in different shades. The DNA is omitted for clarity. (B) View of

608 the structure displayed in A rotated 90°.

609

610 Figure 2. Characterization of the replication defects of class II IN mutant viruses.

611 (A) Immunoblot analysis of Gag and Gag-Pol products in cell lysates and virions. HEK293T cells

612 were transfected with proviral HIV-1NL4-3 expression plasmids carrying pol mutations encoding

613 for the indicated IN substitutions. Cell lysates and purified virions were harvested two days post

614 transfection and analyzed by immunoblotting for CA, IN and, in the case of virions, RT.

615 Representative image of one of four independent experiments is shown. (B) Infectious titers of

616 WT or IN mutant HIV-1NL4-3 viruses in cell culture supernatants were determined on TZM-bl

617 indicator cells. Titer values are expressed relative to WT (set to 1). Columns show average of

618 five independent experiments (open circles) and error bars represent standard deviation (****P

619 < 0.0001, by one-way ANOVA with Dunnett’s multiple comparison test). (C) Relative quantity of

620 reverse-transcribed HIV-1 DNA in MT-4 target cells infected with HIV-1NL4-3 at 6 hpi. Quantities

621 of vDNA are expressed relative to WT (set to 1). Columns show average of three independent

622 experiments (open circles) and error bars represent standard deviation (****P < 0.0001, by one-

623 way ANOVA with Dunnett’s multiple comparison test). (D) Representative growth curve of HIV-

624 1NL4-3 IND116N viruses trans-complemented with class II mutant IN proteins in cell culture. Y-axis

625 indicates fold increase in virion yield over day 0 as measured by RT activity in culture

626 supernatants. HIV-1NL4-3 IND116N viruses that were trans-complemented with WT IN, class II

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627 mutant INs, IND116N, or an empty vector are denoted as red, black, dark blue and light blue lines

628 respectively. Representative plot from one of three independent experiments. (E) Fold increase

629 in virions in culture supernatants at 4 dpi, as measured by RT activity in culture supernatants.

630 Trans-complementation of the HIV-1NL4-3 IND116N virus with mutant IN molecules restored particle

631 release to levels comparable to WT IN (red), partially restored particle release (gray) or could

632 not restore particle release (blue). Columns show average of three independent experiments

633 (open circles) and error bars represent standard deviation (*P < 0.05 and **P < 0.01, by paired t

634 test between individual mutants and WT).

635

636 Figure 3. Class II IN substitutions prevent IN binding to the vRNA genome in virions.

637 (A) Analysis of the levels of packaged viral genomic RNA in WT and IN mutant HIV-1NL4-3

638 virions. vRNA extracted from purified virions was measured by Q-PCR. Data was normalized to

639 account for differences in particle yield using an RT activity assay. Normalized quantities of

640 vRNA are expressed relative to WT (set to 1). Columns show the average of three-four

641 independent experiments (open circles) and error bars represent standard deviation (ns, not

642 significant, by one-way ANOVA). (B) Representative autoradiogram of IN-RNA adducts

643 immunoprecipitated from WT or IN mutant HIV-1NL4-3 virions. The amount of immunoprecipitated

644 material was normalized such that equivalent levels of WT and mutant IN proteins were loaded

645 on the gel, as also evident in the immunoblots shown below. Levels of IN and CA in input virion

646 lysates is shown in the lower immunoblots. Data is representative of three independent

647 replicates.

648

649 Figure 4. Multimerization properties of class II IN mutants.

650 (A) Immunoblot analyses of IN multimers in virions. Purified WT or IN mutant HIV-1NL4-3 virions

651 were treated with 1 mM EGS, and virus lysates analyzed by immunoblotting using antibodies

652 against IN following separation on 6% Tris-acetate gels. Position of monomers (M), dimers (D),

26 bioRxiv preprint doi: https://doi.org/10.1101/2019.12.18.881649; this version posted December 19, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

653 and tetramers (T) are indicated by arrows. Representative image of one of three independent

654 experiments is shown. (B) SEC profiles of 10 µM INs by Superdex 200 10/300 GL column. X-

655 axis indicates elution volume (mL) and Y-axis indicate the intensity of absorbance (mAU).

656 Tetramers (T), Dimers (D) and Monomers (M) are indicated. Representative chromatograms

657 from two independent analyses are shown. (C) Analysis by Alpha screen assay of 100 nM WT

658 IN monomers, dimers, and tetramers binding to biotinylated TAR RNA after separation by SEC.

659 Graphed data is the average of three independent experiments and error bars indicate standard

660 deviation. (D) Analysis of 100 nM WT or mutant INs binding to biotinylated TAR RNA by Alpha

661 screen assay. Graphed data is the average of three independent experiments and error bars

662 indicate standard deviation.

663

664 Figure 5. Analysis of class II IN mutant virion morphologies viruses by TEM.

665 (A) Representative TEM images of WT, K14A, N18I, K34A, E87A, E96A, W108R, F185K,

666 R187A, L241A, L242A, R262A/R263A, and ∆RT-IN HIV-1NL4-3 virions. Magnification is 30,000x

667 (scale bar, 100 nm). Black arrows indicate mature particles containing conical or round cores

668 with associated electron density; triangles indicate eccentric particles with electron dense

669 material situated between translucent cores and the viral membrane; diamonds indicate

670 immature particles. (B) Quantification of virion morphologies. Columns show the average of two

671 independent experiments (more than 100 particles counted per experiment) and error bars

672 represent standard deviation.

673

674 Figure 6. Biochemical analysis of class II IN mutant virus particles.

675 (A) Immunoblot analysis of sedimentation profiles of IN in WT or IN mutant virions. Purified HIV-

676 1NLGP virions were analyzed by equilibrium density centrifugation as detailed in Materials and

677 Methods. Ten fractions collected from the top of the gradients were analyzed by immunoblotting

678 using antibodies against IN. Representative images from one of four independent experiments

27 bioRxiv preprint doi: https://doi.org/10.1101/2019.12.18.881649; this version posted December 19, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

679 are shown. (B) Quantitation of IN signal intensity in immunoblots as in (A) are shown. Profile of

680 WT virions is denoted in black, IN mutants that led to bimodal IN distribution are shown in red

681 and others are shown in grey. Graphed data is the average of two independent experiments and

682 error bars indicate the range. (C) Representative immunoblot analysis of sedimentation profile

683 of CA in WT virions and quantitation of CA signal intensity in immunoblots are shown. Profile of

684 WT virions is denoted in black, IN mutants that led to bimodal IN distribution are shown in red

685 and others are shown in grey. Graphed data is the average of two independent experiments and

686 error bars indicate the range.

687

688 Figure 7. Premature loss of vRNA and IN from class II IN mutant viruses upon infection of

689 target cells.

690 (A) Locations of the class II IN substitutions K34A, E87A, V165A and R262A/R263A displayed

691 on a single IN monomer within the context of the HIV-1 IN tetramer intasome structure (PDB

692 5U1C.) Substitutions are color coded based on whether they putatively caused mislocalization

693 of IN in virions (black) or not (blue.) (B-E) PgsA-745 cells were infected with WT or IN mutant

694 HIV-1 virions and fates of viral core components were analyzed 2 hpi. Fractions were analyzed

695 for the presence of CA (B) and IN (C) by immunoblotting and for RT activity (D) and vRNA (E)

696 by Q-PCR. Immunoblots are representative of three independent experiments. Graphed data in

697 (D) and (E) is the average of three independent experiments with error bars indicating standard

698 deviation (*P < 0.05 and **P < 0.01, by repeated measures one-way ANOVA.) (F)

699 Representative images of pgsA-745 cells infected with WT or IN mutant HIV-1NL4-3 viruses 0 and

700 2 hpi. Cells were stained for vRNA (green) and nuclei (blue) as detailed in Materials and

701 Methods. (G) Fraction of viral RNA remaining after 2 and 6 hpi compared to the quantity

702 measured at 0 hpi. MT-4 cells were synchronously infected with VSV-G pseudotyped HIV-1NL4-3

703 viruses and at each timepoint samples of infected cultures were taken for analysis. Viral RNA

28 bioRxiv preprint doi: https://doi.org/10.1101/2019.12.18.881649; this version posted December 19, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

704 levels in samples were measured by Q-PCR and normalized to the levels of GAPDH mRNA.

705 Data points are the average of five independent experiments with error bars indicating standard

706 error of the mean.

707

708 Figure 8. Model depicting how class II IN mutants exert their effects on HIV-1 replication

709 Figure S1. Characterization of the replication defects of class II IN mutant viruses.

710 (A) Reverse-transcriptase activity measured in HIV-1NL4-3 virion lysates. For each repetition RT

711 activities for the IN mutants are expressed relative to the WT (set to 1.) Columns show average

712 of two independent experiments (open circles) and error bars represent standard deviation

713 (****P < 0.0001, ***P < 0.001, **P < 0.01, and *P < 0.05, by unpaired t test between individual

714 mutants and WT.) (B) Representative immunoblot analysis of Vpr-IN fusion constructs in cell

715 lysates. HEK293T cells were co-transfected with the HIV-1NL4-3 IND116N proviral plasmid along

716 with Vpr-IN expression plasmids encoding for the indicated IN substitutions or an empty vector

717 control. Expression of Vpr-IN constructs in cell lysates was detected using an anti-IN antibody.

718

719 Figure S2. Premature loss of vRNA and IN from class II IN mutant viruses upon infection

720 of target cells.

721 (A) Representative images of uninfected pgsA-745 cells and cells infected with WT HIV-1NL4-3

722 viruses at 0 hpi. Cells were fixed and stained for vRNA (green) and nuclei (blue). (B)

723 Representative images of pgsA745 cells infected with IN mutant HIV-1NL4-3 viruses 0 and 2 hpi.

724 Cells were fixed and stained for vRNA (green) and nuclei (blue). (C) Quantification of vRNA

725 remaining in cells infected with WT or IN mutant HIV-1NL4-3 viruses at 2 hpi. Values are the

726 percent of vRNA remaining at 2 hpi compared to at 0 hpi. Columns show average of three

727 independent experiments (open circles) and error bars represent standard deviation (*P < 0.05

728 and **P < 0.01, by one-way ANOVA with Dunnett’s multiple comparison test.) (D) Fraction of

29 bioRxiv preprint doi: https://doi.org/10.1101/2019.12.18.881649; this version posted December 19, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

729 viral RNA remaining after 2 and 6 hpi compared to the quantity measured at 0 hpi. MT-4 cells

730 were synchronously infected with VSV-G pseudotyped HIV-1NL4-3 viruses and incubated in the

731 presence of 50 mM ammonium chloride for 6 hrs. At each timepoint samples of infected cultures

732 were taken for analysis and levels of viral RNA in samples were measured by Q-PCR and

733 normalized to the levels of GAPDH mRNA. Data points are the average of three independent

734 experiments with error bars indicating standard error of the mean.

735

736 ACKNOWLEDGEMENTS: We thank Dr. Michael Malim for providing the anti-IN monoclonal

737 antibody and members of the Tolia lab for assisting in PyMol analysis. This study was supported

738 by grants NIH grants P50 GM103297 (the Center for HIV RNA Studies) and GM122458

739 to SBK, AI143389-F31 fellowship to JE, R01 AI062520 to MK and SBK, U54 AI150472 to MK

740 and AE, AI070042 to AE.

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35 bioRxiv preprint doi: https://doi.org/10.1101/2019.12.18.881649; this version posted December 19, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

A CTD Q214L/Q216L L242A K215A/K219A K258A R228A V260E K236E R262A/R263A L241A R269A/K273A

NTD CCD H12N E87A K14A E96A N18I Y99A K34A K103E W108R V165A F185K K186A K186E R187A K188E 90° R199A B CCD NTD E87A E96A H12N Y99A K14A K103E N18I W108R K34A V165A F185K K186A CTD K186E Q214L/Q216L L242A R187A K215A/K219A K258A K188E R228A V260E R199A K236E R262A/R263A L241A R269A/K273A

Figure 1 D B A Intermediates Processing

0.001 Relative infectivity 10000 0.01

Fold Change 1000 0.1 100 10 0.1 10 1 Gag-Pol Gag-Pol 1

WT 0 H12N (which wasnotcertifiedbypeerreview)istheauthor/funder,whohasgrantedbioRxivalicensetodisplaypreprintinperpetuity.Itmade K14A bioRxiv preprint kD

N18I 28 39 51 28 39 51 64 K34A

E87A Particle Release 2 Particle Release E96A

Y99A WT Days K103E H12N W108R

V165A K14A doi: 4 F185K

Viral Titer N18I K186A IN Mutant https://doi.org/10.1101/2019.12.18.881649 K186E K34A R187A ****

K188E E87A 6 Q214L/Q216L R199A K215A/K219A E96A Y99A H12N Y99A E96A E87A K34A K14A K186A V165A K103E N18I D F185K Vector W W108R

1 R228A T 16N K103E K236E L241A W108R L242A V165A K258A R262A/R263A V260E R262A/R263A K215A/K219A Q214L/Q216L V260E K258A K236E K188E K186E L242A L241A R269A/K273A R228A R199A R187A R269A/K273A F185K

K186A available undera K186E R187A E C

0.001 pg/mL Relative value WT 0.01 0.0 0.5 1.0 1.5 100 0.1 10

1 K188E WT + Nev CC-BY 4.0Internationallicense WT ; PCR3.1WT R199A this versionpostedDecember19,2019. D1

D1 ** 16N 16N

** H12N Q214L/Q216L H12N K14A K14A ** K215A/K219A N18I N18I K34A K34A ** R228A E87A E87A ** E96A K236E E96A * Y99A Y99A K103E L241A K103E W108R W108R L242A V165A R

V165A ** Particle Release F185K T Pr

IN Mutant K258A F185K K186A IN Mutant K186A * K186E V260E **** oduc K186E * R187A R187A R262A/R263A *

K188E . Q214L/Q216L K188E Q214L/Q216L R199A t R296A/K273A

K215A/K219A s R199A K215A/K219A ΔIN

* R228A D116N R228A * K236E The copyrightholderforthispreprint α-RT (virion) α-CA (virion) α-CA (cell) α-IN (virion) α-IN (cell) α-CA (cell) K236E ** L241A L241A L242A

L242A * K258A R262A/R263A

Figure 2 K258A R262A/R263A * R269A/K273AV260E

V260E * R269A/K273A ** * bioRxiv preprint doi: https://doi.org/10.1101/2019.12.18.881649; this version posted December 19, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

A B L A A

Viral RNA Packaging A A

16N 16N

3 IN ns IN K34A E87A E96A V165A K186A R187A K14A R199A W T D 1 W T K188E 4/56 !"#$ %&'$ %()$ *")+$ !"&)$ ,"&'$ !"&&% ,"(($ -."#/0-.")/ !.1)% /.#.$ !.+&$ ,.).$0,.)1$ D 1 Q214L/Q216 K236E L241 L242 K258 A R262A/R263 R269A/K273 4/56 !"#$ %&'$ %()$ *")+$ !"&)$ ,"&'$ !"&&% ,"(($ -."#/0-.")/ !.1)% /.#.$ !.+&$ ,.).$0,.)1$ @AB @ABkD kD

64 2 64 #( #( 51 51 1 1& 1& 347,4$ 347,4$ 39 39 89:;<=>=? 89:;<=>=? IN:RNA Relative value (vRNA/RT ) 0

WT 16N K14AK34AE87AE96A α-IN D1 V165A K188E K236EL241AL242A 34 34 IP delta IN K186AR187A R199A K258A Q214L/Q216LK215A/K219A R262A/R263AR269A/K273A 2$ 2$ α-IN Lysate IN Mutant α-CA

Figure 3 B A C 210 kDa 28 41 55 71 K188E IN WT IN (which wasnotcertifiedbypeerreview)istheauthor/funder,whohasgrantedbioRxivalicensetodisplaypreprintinperpetuity.Itmade bioRxiv preprint

WT T T ΔIN D D K14A doi: M K34A M

E87A https://doi.org/10.1101/2019.12.18.881649 E96A V165A K186A K34A IN K236E IN WT ΔIN available undera R187A T K188E D

D R199A

M Q214L/Q216L K236E CC-BY 4.0Internationallicense ; this versionpostedDecember19,2019.

WT ΔIN L241A R262A/R263A IN

E96A IN K258A R262A/R263A R296A/K273A T T D116N D D . M M D T The copyrightholderforthispreprint Figure 4 bioRxiv preprint doi: https://doi.org/10.1101/2019.12.18.881649; this version posted December 19, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

A WT K14A N18I K34A E87A

E96A W108R F185K R187A L241A

L242A R262A/R263A ΔRT-IN

100 nm

B Virion Morphology 100 **** Mature 80 Immature Eccentric 60

40

20 EM Morphology (% )

0

WT K14A N18I K34A E87A E96A W108R F185K R187A L241A L242A RT-IN

R262A/R263A IN Mutant

Figure 5 bioRxiv preprint doi: https://doi.org/10.1101/2019.12.18.881649; this version posted December 19, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

A B 0.5 Fraction WT WT K34A 1 2 3 4 5 6 7 8 9 10 E87A 0.4 E96A WT V165A K186A K34A 0.3 R187A K188E R199A E87A Q214L/Q216L 0.2 K236A E96A L241A Fraction of total IN signa l K258A 0.1 V165A R262A/R263A R269A/K273A

K186A 0.0 1 2 3 4 5 6 7 8 9 10 R187A Fraction C K188E Fraction 1 2 3 4 5 6 7 8 9 10 R199A WT

1 Q214L/Q216L WT K34A K236A E87A E96A V165A L241A signa l 0.1 K186A R187A K258A K188E R199A R262A/R263A Q214L/Q216L K236A 0.01 L241A

R269A/K273A Fraction of total C A K258A R262A/R263A α-IN R269A/K273A

0.001 1 2 3 4 5 6 7 8 9 10 Fraction

Figure 6 bioRxiv preprint doi: https://doi.org/10.1101/2019.12.18.881649; this version posted December 19, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

CTD CTD A R262A/R263A R262A/R263A 90° NTD NTD K34A K34A CCD CCD E87A E87A V165A V165A

B Fraction C Fraction 1 2 3 4 5 6 7 8 9 10 1 2 3 4 5 6 7 8 9 10 WT WT K34A K34A E87A E87A V165A V165A R262A/R263A R262A/R263A R269A/K273A R269A/K273A CA IN D E 0.4 RT Activity 0.4 Viral RNA

WT * WT 0.3 0.3 K34A K34A E87A E87A V165A V165A 0.2 0.2 ** R262A/R263A * R262A/R263A R269A/K273A R269A/K273A Relative copie s Relative copie s 0.1 0.1

0.0 0.0 1 2 3 4 5 6 7 8 9 10 1 2 3 4 5 6 7 8 9 10 Fraction number Fraction number

F 0 hr 2 hr G Viral RNA 2

1 WT

0.5 WT K34A E87A V165A 0.25

Fraction remainin g R262A/R263A

0.125 0 2 6 Time (hr)

K34A

20 μm Figure 7 bioRxiv preprint doi: https://doi.org/10.1101/2019.12.18.881649; this version posted December 19, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

WT HIV-1 Class II IN Mutant HIV-1

IN binds vRNA IN cannot bind vRNA

ii. IN multimerization defect iii. Direct IN-RNA i. IN reduced binding defect

viral RNA

IN

CA

Mature virus particle Eccentric virus particle

vRNA and IN lost, reverse-transcription not completed Sucessful reverse-transcription Figure 8 bioRxiv preprint doi: https://doi.org/10.1101/2019.12.18.881649; this version posted December 19, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

!"#$%&'(&)*&$%+%$,&-.&+-/-0.,& )*&123".3& )*&,-4."$&567& 89& )*&123".3& )*&,-4."$&567& 89& !"# $%%# &'(# )$*+,# +-./# $+.0# 1$/&# &2# &'(# 3$*4(# -0.0# /0.5# )$0(# 0%.5# $%.-# )$**,# 05.*# /+.-# &$*6# $+.4# -.$# 3$77(# 0$.$# $7./# )-0(# -/.+# +.7# 8/$09'8/$+9# -$.4# +.-# ,*4(# -$.0# )/$5(')/$7(# 04.4# /%.0#

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

!

Table 1 bioRxiv preprint doi: https://doi.org/10.1101/2019.12.18.881649; this version posted December 19, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

A RT Activity 2.0

1.5

1.0 Relative valu e

0.5

0.0 IN WT 16N H12NK14AN18IK34AE87AE96AY99A K103EW108RV165AF185KK186AK186ER187AK188ER199A R228AK236EL241AL242AK258AV260E D1

Q214L/Q216LK215A/K219A R262A/R263AR269A/K273A IN Mutant B

kDa WT Vector D116N H12N K14A N18I K34A E87A E96A Y99A K103E W108R V165A F185K K186A WT K186E R187A K188E R199A Q214L/Q216L K215A/K219A R228A K236E L241A L242A K285A V260E R262A/R263A R296A/K273A 51 α-IN 39

Figure S1 bioRxiv preprint doi: https://doi.org/10.1101/2019.12.18.881649; this version posted December 19, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

A C Infected Uninfected Viral RNA 100 * **

80

60

40

10 μm Percent remainin g

20 B 0 hr 2 hr 0

WT K34A E87A V165A

IN Mutant R262A/R263A E87A D Viral RNA (+NH4Cl) 2

1

0.5 WT K34A E87A V165A Fraction remainin g V165A 0.25 R262A/R263A

0.125 0 2 6 20 μm Time (hr)

R262A/ R263A

20 μm

Figure S2